Beyond Olive Oil: Active Components and Health Aspects of Some

Chapter 13

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Beyond Olive Oil: Active Components and Health Aspects of Some Less Studied Mediterranean Plant Products Nick Kalogeropoulos,* Antonia Chiou, Andriana C. Kaliora, Vaios T. Karathanos, and Nikolaos K. Andrikopoulos Department of Science of Dietetics - Nutrition, Harokopio University, 17671 Athens, Greece *E-mail: [email protected]

Current advances on health benefits of Mediterranean diet components considered foods or functional food items, namely legumes, aromatic plants, Chios mastic, and currants, provide significant guidance for further research and for industries in developing nutraceutical products. Cooked legume polyphenols (13.4-25.9 mg gallic acid eq (GAE) /100g) – mainly flavonoids in lentils and chickpeas, phenolic acids in the others – and triterpenic acids (0.29-8.55 mg/100g) inhibit LDL oxidation in vitro. The infusions of 12 aromatic plants (polyphenolic content between 5.3-159.2 mg GAE/cup) exhibit potent antiradical activity (7.7-201.3 mg Trolox® eq/cup) and ferric ion reducing antioxidant power (FRAP) values between 0.5-66.5 mg ascorbic acid eq/cup. Terpene rich Chios mastic exhibits potent antioxidant, anti-inflammatory and in vivo immunoregulating activity in inflammatory bowel disease. Polyphenol rich currants (151-246 mg GAE/100g – mainly phenolic acids) exhibit antiradical, LDL antioxidant and mononuclear cell cytoprotective activity. Further, currants induce death and exhibit antiproliferative and anti-inflammatory effect in gastric adenocarcinoma cells.

© 2012 American Chemical Society In Emerging Trends in Dietary Components for Preventing and Combating Disease; Patil, B., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.


Introduction. World Mortality Pattern Changes According to the 2008 WHO health report (http://www.who.int/whr/2008/ whr08_en.pdf), there is a striking shift in distribution of death and disease from younger to older ages and from infectious, perinatal and maternal causes to noncommunicable diseases– including depression, diabetes, cardiovascular disease and cancers (1). There is increasing evidence that oxidative stress is implicated in the pathogenesis of many inflammatory and degenerative diseases (2, 3). Oxidative stress occurs when the oxidant-antioxidant balance becomes too favourable to prooxidants. In modern Western medicine, the balance between antioxidation and oxidation is believed to be a critical concept maintaining a homeostasis (4, 5). Current hypotheses favour the idea that regulating oxidative stress can provide clinical benefits. As dietary habits are associated with several chronic diseases -including cardiovascular disease, cancer and immune dysfunction- the nutritional approach could be a useful tool for lowering oxidative stress. Indeed, there are now well established nutritional recommendations for prevention of cancer, atherosclerosis and other chronic diseases. The traditional Mediterranean diet of Greece is considered beneficial against chronic and degenerative diseases (6). It is characterized by reduced intake of saturated lipids and animal proteins, and high intake of olive oil, vegetables, fruits, grains, potatoes, nuts, legumes which provide significant amounts of phytochemicals. Phytochemicals are bioactive plant components, which do not provide energy, usually exhibit antioxidant action, and exert beneficial effects for human health (cardioprotective, anti-inflammatory, anti-tumour etc). Examples of phytochemicals are tocopherols, polyphenols, terpenes, carotenoids, ascorbic acid etc. Although the most characteristic feature of Mediterranean type diets is the consumption of high amounts of olive oil, other less known Mediterranean plant products exhibit potential health effects due to their significant content in bioactive microconstituents and obviously contribute to the overall beneficial effects of such diets. This chapter presents the main phytochemicals and the antioxidant capacity of some less studied Mediterranean plant foods, in the form they are actually consumed, and establishes their potential health benefits, in vitro and/or in vivo. The plant foods selected for this study are (a) cooked dry legumes, (b) infusions of aromatic-medicinal plants, (c) Pistachia Lentiscus resin (Chios mastic gum), (d) currants and sultanas (dried vine fruits).

Legumes Legumes have long been associated with longevity food cultures. For example, the Japanese eat soy, tofu, natto and miso, the Swedes eat brown beans and peas and the Mediterraneans eat lentils, chickpeas and white beans (7–9). Legumes contain almost twice the amount of protein compared to cereal grains, while they are low in fat, and rich in complex hydrocarbons and minerals (10). 238 In Emerging Trends in Dietary Components for Preventing and Combating Disease; Patil, B., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.


Furthermore, legumes exhibit lower glycaemic indices compared to other starchy foods and contain phytosterols, natural antioxidants and bioactive carbohydrates, which are considered potentially beneficial for human health (11–13). Both epidemiological and clinical intervention studies showed that legume consumption is inversely associated with the risk of coronary heart disease, obesity, and type II diabetes mellitus (14–17), while it results in lower LDL and higher HDL cholesterol levels (18, 19). Nowadays legumes gain increasing attention as functional food items and increase in their consumption within the Western diet is recommended (20, 21). It should be noted hereby that legumes contain certain antinutritional factors, among them trypsin inhibitors, α-galactosides, and phytic acid (22–24), which are normally diminished during legume cooking. With the exception of soybean, there is relatively little information on the phytochemical content of cooked legumes (25). To investigate the phytochemical content of dry cooked legumes Kalogeropoulos et al (26) prepared several kinds of legumes that are often consumed in Greece and other Mediterranean countries, by fisrt soaking and then boiling them in tap water. The obtained cooked legumes were subsequently homogenised, freeze-dried and analysed for phytosterols, tocopherols, simple polyphenols and triterpenic acids (26). In addition, in the same cooked legumes total phenolics were determined; DPPH• radical scavenging activity and ferric ion reducing antioxidant power (FRAP) were also assessed. Finally, the potential of the cooked legumes to inhibit human LDL oxidation in vitro was tested. The results obtained for seven kinds of legumes, namely broad beans, chickpeas, yellow split peas, small lentils and 3 varieties of white beans (medium sized, giant, and elephant beans) are presented and discussed.

Phytochemicals in Cooked Legumes Tocopherols and Phytosterols Tocopherols are considered as very effective lipid phase antioxidants, acting as peroxyl radical scavengers that terminate chain reactions in membranes and lipoprotein particles (27). Kalogeropoulos et al (26) reported tocopherols – mainly β- and γ- homologues – to be present at concentrations ranging from 0.26 - 0.36 mg/100g in white beans and broad beans to 0.82 and 1.78 mg/100g in lentils and chickpeas, respectively (26), their values being one order of magnitude lower than the respective reported by other researchers for uncooked legumes (11, 28), due to soaking and boiling. Phytosterols were also present in the cooked legumes at concentrations that ranged from 22.9 – 48.9 mg/100g in lentils and chickpeas, respectively (26), with β-sitosterol predominating in all cases. As a result of soaking/cooking the phytosterols present in the cooked legumes were 4-8 times lower than the values reported for uncooked legumes (28), still representing the 15.8-24.4% of phytosterols daily dietary intake (26). 239 In Emerging Trends in Dietary Components for Preventing and Combating Disease; Patil, B., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.


Simple Polyphenols Polyphenols are known to exert antioxidant anti-inflammatory and antimicrobial action, while accumulating literature data indicate that they protect body tissues against oxidative stress; they have been shown to be protective in vitro against several types of cancer, such as breast, prostate, skin, and colon cancer (29). There is now emerging evidence that their metabolites exert modulatory effects in cells through selective actions on different components of the intracellular signalling cascades, vital for cellular functions such as growth, proliferation and apoptosis (25). There are several reports on the presence of polyphenols in uncooked legumes, but relatively fewer studies on the respective content of cooked or processed legumes, as reviewed recently by Amarowizc and Pegg (11). Phenolic acids and flavonoids are the commonly reported simple phenolics in legumes (11, 13, 30–34). Kalogeropoulos et al. (26) reported the presence of both phenolic acids and flavonoids in several kinds of cooked legumes. Among the phenolic acids caffeic acid, cinnamic acid, o-coumaric acid, p-coumaric acid, gallic acid, ferulic acid, p-hydroxybenzoic acid, phloretic acid, protocatechuic acid, sinapic acid, and vanillic acid were present in the majority of cooked legumes (26) studied. Among the flavonoids determined in cooked legumes, catechin and epicatechin predominated followed by chrysin, genistein, quercetin and kaempferol (26). The same flavonoids together with apigenin, luteolin, daidzein and coumestrol are present in raw, cooked or germinated leguminous seeds and their extracts, according to a recent review by Amarowicz and Pegg (11). Lentils have been reported to be consistently rich in flavonoids in raw (35) or cooked form (26).

Triterpenic Acids Triterpenic compounds are common constituents of plants. They are relatively non-toxic and possess pharmacological properties exerting anti-inflammatory, hepatoprotective, antitumour, antiviral, anti-HIV, antimicrobial, antifungal, antidiabetic, gastroprotective and antihyperlipidemic action. The triterpenic acids oleanolic, ursolic and maslinic were present in the seven cooked legumes studied in concentrations ranging from 0.34-8.55 mg/100g in medium beans and chick peas, respectively. Oleanolic acid was present in all cooked samples; ursolic acid was present in broad beans, chick peas and yellow split peas, while chick peas and lentils contained in addition maslinic acid. Total Phenolic Content and Antioxidant Activities of Cooked Legumes The total phenolic content, together with the DPPH• free radical scavenging capacity and the FRAP values of the cooked legumes studied are presented in Figure 1. The total phenolic content of the legumes studied ranged from 13.4 mg of gallic acid equivalents (GAE) per 100g in medium beans to 25.9 mg GAE/100g in small lentils. These values are generally lower than those reported for uncooked legumes. The obvious explanation for this decrement is the soaking and boiling 240 In Emerging Trends in Dietary Components for Preventing and Combating Disease; Patil, B., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.


of legumes, which results in partial leaching and thermal/oxidative destruction of phenolics, in agreement with previous reports on the effect of soaking and cooking on total phenolic contend of several types of legumes (33, 36–39). The DPPH scavenging activity of cooked legumes ranged from 0.24 mmol Trolox equivalents (TE) /100g in broad beans to 2.1 mmol TE/100g in lentils, while the FRAP values ranged from 1.3 µmol ascorbic acid equivalents (AAE) /100g in elephant beans to 7.1 µmol AAE/100g in lentils (Figure 1), values that are lower than those reported by Xu and Chang (40) for uncooked peas, chickpeas, lentils, kidney beans and black beans.

Figure 1. Total phenolic content (mg GAE, divided by 10), free radical scavenging activity (mmol TE) and FRAP reducing potential (µmol AAE) of 100 g cooked legumes; GAE stands for gallic acid equivalents;TE stands for Trolox® equivalents; AAE stands for ascorbic acid equivalents.

In Vitro Inhibition of LDL Oxidation by Cooked Legumes Human low density lipoprotein (LDL) particle is recognized as being highly vulnerable to oxidation by reactive oxygen species (ROS), leading to the formation of oxidized LDL (oxLDL) which is strongly implicated in atherogenesis (41, 42). The potential of cooked legumes to inhibit the Cu2+ induced LDL oxidation, was tested in vitro. For this purpose, LDL oxidation in the presence or absence of cooked legumes’ extracts was followed, and the respective lag phase times were compared. Amounts of LDL and legume extracts used were calculated to represent the LDL in an adult’s circulation –with blood volume of 4L and LDL concentration equal to 150 mg/dL (43) –and the extract obtained from one exchange (0.5 cup = 115 g) of cooked legumes, respectively. As shown in Figure 2, all samples tested exhibited inhibitory activity against LDL oxidation with the relative effectiveness 241 In Emerging Trends in Dietary Components for Preventing and Combating Disease; Patil, B., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.


increasing in the order: split peas < broad beans < giant beans < elephant beans < chick peas < medium beans < lentils. Lentils have been reported to exhibit higher inhibitory activity against LDL oxidation compared to other food legumes (40). The inhibitory ability of the legumes studied, correlated better with flavonoids and free radical scavenging capacity, while it was not correlated with phytosterols, squalene, tocopherols or terpenic acids content (data not shown). Flavonoids have been found to exert anti-inflammatory activity (44) and to act against LDL oxidation, their antioxidant capacity being related to their chemical structures (45).

Figure 2. Antioxidant activities of cooked legume extracts on Cu2+- induced LDL oxidation based on detection of conjugated dienes. The values represent the prolongation rates relative to control, from assays performed in duplicate. The amounts of LDL and legume extracts were calculated to represent the LDL in an adult’s circulation and one cup (115g) of cooked legumes, respectively.

Infusions of Mediterranean Aromatic and Medicinal Plants Herbal infusions are considered among the significant sources of dietary polyphenols (46), exerting antioxidant activity which is mainly attributed to the presence of essential oils and polar phenolic compounds. Due to geomorphological characteristics, the flora of the Mediterranean basin presents high biodiversity with many endemic plants. The aromatic and medicinal plants of the Mediterranean have a long tradition of use as folk remedies and culinary herbs since antiquity. In Greece, herbs are consumed in the form of infusions prepared by one herb alone or –as in the case of St John’s wort- in mixture with other aromatic plants. 242 In Emerging Trends in Dietary Components for Preventing and Combating Disease; Patil, B., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.


The composition and antioxidant activity of essential oils and extracts of aromatic herbs obtained by different solvents have been extensively studied. On the contrary, little is known about the presence of antioxidants in herbs’ infusions and decoctions, which we actually consume (47, 48). For this reason infusions of 12 aromatic plants, purchased from the island of Crete - except St John’s wort which was purchased from Central Macedonia, Northern Greece, were prepared by boiling 3 g of dried aerial parts of the plants for 3-4 minutes in 200 mL (one cup) tap water. The plants selected (Table 1) are members of the Lamiaceae family, with the exception of Hypericum perforatum –Cluciaceae- and Matricaria chamomilla –Asteraceae. The infusions obtained were filtered, freeze-dried and analyzed for total phenolics, DPPH• radical scavenging activity and FRAP. In addition, simple polyphenols and terpenic acids were determined by GC/MS as previously reported (49).

Table 1. Common and scientific names, and distribution of the selected aromatic plants Common name

Scientific Name

Distribution

Cretan marjoram

Origanum microphyllum

Endemic, Crete island

Rosemary

Rosmarinus officinalis

Native, Mediterranean

Cretan dittany, dictamnus

Origanum dictamnus

Endemic, Crete island

Pink savory

Satureja thymbra


Thyme

Thymus vulgaris

Southern Europe

Marjoram

Origanum Majorana

Indigenous, Mediterranean

Oregano

Origanum vulgare

SW Eurasia, Mediterranean

Greek mountain tea

Sideritis syriaca

Endemic, Greece

St John’s wort

Hypericum perforatum

Worldwide

Sage

Salvia officinalis


Pennyroyal

Mentha pulegium

Europe

Chamomile

Matricaria chamomilla

Europe

All the infusions contained significant amounts of phenolics, and exhibited free radical scavenging activity and reducing potential against Fe3+ (Table 2). Total phenolic content ranged from 5.3-159.2 mg GAE per cup (=200 mL), comparable to the values of 88 - 184 mg GAE per cup reported for Greek herbal infusions (48). DPPH• radical scavenging capacity ranged from 0.03-1.72 mmol TE per cup, and FRAP ranged between 0.5-66.5 mg AAE per cup (Table 2). In all cases the lower 243 In Emerging Trends in Dietary Components for Preventing and Combating Disease; Patil, B., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.

values were observed for the infusion of rosemary (R. officinalis) and the higher for that obtained from St John’s wort (H. perforatum).

Table 2. Total phenolic content, free radical scavenging activity and reducing potential of the aromatic plants’ infusions Total phenolics (mg GAE/cup*)

DPPH• scavenging activity (mmol TE/cup)

Ferric reducing antioxidant power, FRAP, (mg AAE/cup)

O. microphyllum

17.1±2.1

0.21±0.01

4.7±0.17

R. officinalis

5.3±4.2

0.03±0.02

0.5±0.07

O. dictamnus

29.7±5.2

0.57±0.02

16.5±0.72

S. thymbra

52.6±1.3

0.52±0.01

17.0±1.4

T. vulgaris

32.5±5.5

0.28±0.02

6.8±0.15

O. majorana

59.2±1.3

0.60±0.01

19.1±1.1

O. vulgare

61.9±4.8

0.80±0.02

29.1±2.7

S. syriaca

21.5±3.8

0.21±0.02

6.7±0.08

159.2±11.2

1.72±0.07

66.5±5.0

S. officinalis

18.7±1.8

0.21±0.01

4.7±0.41

M. pulegium

61.9±2.0

0.72±0.01

20.2±0.75

M. chamomilla

17.7±2.5

0.32±0.01

7.5±0.39


Aromatic plant

H. perforatum

Results are means ± SD (n=3); * 1cup = 200 mL; GAE stands for gallic acid equivalents; TE stands for Trolox® equivalents; AAE stands for ascorbic acid equivalents.

Simple polyphenols content, quantified by GC/MS, ranged from 0.14-38.0 mg/cup in R. officinalis and H. perforatum, respectively, and were mainly phenolic acids and flavonoids; Among 18 phenolic acids tested, 13 were present in more than 5 infusions, namely p-hydroxybenzoic acid, caffeic acid, cinnamic acid, chlorogenic acid, o-coumaric acid, p-coumaric acid, ferulic acid, gallic acid, 3,4-dihydroxy-phenylacetic acid, protocatechuic acid, syringic acid, sinapic acid, and vanillic acid. Total phenolic acids concentrations ranged between 0.12-5.96 mg/cup in R. officinalis and M. chamomilla, respectively (Figure 3). Regarding flavonoids, the aglycons of epicatechin and catechin were present in all infusions, while aglycons of chrysin, genistein, kaempferol, naringenin, and quercetin were present in the majority of them, their summed concentrations ranging from 0.013-36.1 mg/cup in R. officinalis and H. perforatum, respectively (Figure 3). Additionally, the triterpenic acids oleanolic and ursolic were detected in the infusions of O. microphyllum, R. officinalis, O. majorana, O. vulgare, 244 In Emerging Trends in Dietary Components for Preventing and Combating Disease; Patil, B., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.


S. officinalis, and M. pulegium, at concentrations ranging between 0.014-0.11 mg/cup. The results obtained indicate that the consumption of one cup of the infusions studied provide certain amounts of polyphenols and terpenic acids, exhibiting significant antioxidant and radical scavenging activities.

Figure 3. Phenolic acids, flavonoids and terpenic acids present in one cup (200 mL) of the aromatic plants infusions. Note that the scale is logarithmic.

Chios Mastic: A Tear That Pleases, Perfumes, Relieves, and Heals Chios Mastic or “mastiha” is the name of a resinous sap produced from the mastic tree (Pistacia Lentiscus var. Chia). It is a natural, aromatic resin in teardrop shape, falling on the ground in drops from superficial scratches induced by cultivators on the tree’s trunk and main branches with sharp tools. As it drips, this sap appears as a sticky and translucent liquid, which 15-20 days later is solidified into irregular shapes influenced by the area’s weather conditions in summertime that is intense drought and sunlight. After being solidified, it has a crystal form, while its rather bitter taste quickly subsides to leave a distinctive aroma that really makes it unique. That solid product is then harvested and washed by mastic growers, finally providing the natural Chios Mastic. Its colour is initially ivory-like but as time goes by that shade is lost and 12 to 18 months later it changes into yellowish due to oxidation. It is made of hundreds of components, and such multitude probably justifies the multiple uses of Chios Mastic, in the fields of food industry, health and cosmetic care, worldwide. 245 In Emerging Trends in Dietary Components for Preventing and Combating Disease; Patil, B., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.


Chios Mastic has been recognized since ancient times both for its distinctive aroma and its healing properties. Many ancient Greek authors, including Dioscurides and Theophrastus, mentioned Chios Mastic for its healing properties in intestines, stomach and liver. It has been recorded as the first natural chewing gum in the ancient world. Since 1997, Chios Mastic has been characterized as a Product of Protected Designation of Origin (PDO), on the basis of Regulation No. 123/1997 (L0224/24-1-97) of the European Union and it has been registered on the relevant Community List of PDO Products. According to the above regulation, Chios Mastic is protected from the sale of any competitive imitation product whatsoever that would undermine the reputation of the Designation of Origin (http://www.gummastic.gr ). The lentisk is a rather resilient plant with minor demands that is why it grows well on arid, rocky and poor soil. As its roots are spread on the soil’s surface, it can survive in conditions of absolute drought, but can be extremely sensitive to cold and frost. New cultivations are produced from old trees’ branches (grafts) and the old ones are renewed from offshoots or layers. Chios Island is actually identified with mastic and while there are lentisks all over the island, mastic is only produced in the southern part of Chios, in the so-called Mastihohoria or mastic villages, where the climate is especially warm and dry. Besides a longtime tradition, this “uniqueness” is probably due, to certain soil and weather conditions which favor the mastic tree’s cultivation only in Chios and only in this specific part of the isle. The medicinal and pharmaceutical properties of Chios Mastic were well known ever since antiquity. Nowadays, laboratory research and clinical studies have revealed that natural Chios Mastic is gifted with unique beneficial and therapeutic properties, thus confirming what has been historically recorded over the past. The treatment of various gastric malfunctions with Chios Mastic has been documented. Earlier studies showed that mastic gum possesses anti-ulcer activities (50–52). Although the effects on Helicobacter pylori are controversial (53–56), a recent study showed that mastic administration (0.75 mg/day) reduces H. pylori colonization in stomach of infected mice (p≤0.01) (57). In 2002, the hepatoprotective effect of the aqueous extract from the leaves of P. lentiscus tree on CCl4 intoxicated rats was published (58). Furthermore, the plant has been shown to suppress the extent of iron-induced lipid peroxidation in rat liver homogenates (59). Chios Mastic, exported from Chios to all over the world, is the basis for the production of a great variety of mastic products, such as bakery products, sweets, jams, ice-creams, chocolates, chewing gums, candies, beverages, tea, coffee, dairy products, pasta, sauces, liquors, ouzo and wine. In certain areas of Greece, mostly of the Aegean Sea, mastic is often used as a flavouring for Easter sweets and for a delicious ice-cream known as kaimaki, which has an unusual chewy and stringy texture thanks to the addition of Chios Mastic as a thickening agent. Modern Greek chefs have proved that this spice with its unique aromatic, woodand pine-like, exotic taste can go along with almost everything, from tomatoes in a tasteful sauce to white wine and lemon in most delicate sauces, and even to chocolate with which it makes a perfect match. In Lebanon and Syria they make 246 In Emerging Trends in Dietary Components for Preventing and Combating Disease; Patil, B., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.


a sort of traditional mastic-flavoured cheese. For Arabs, mastic is considered as a great luxury for flavouring food, sweets or milk. As a spoon sweet, mastic is served in a particularly traditional way, inside a glass of water, a version known as “ypovryhio” (submarine). It is also used as an ingredient for ointments against burns. Rosin is a derivative of mastic used for the production of surgical stitches, while mastic oil is widely used in perfume and cosmetics industry. Finally, thanks to its quality as a color stabilizer, mastic is used for the production of high grade varnishes. Potential Antiatherogenic Effect of Chios Mastic Effect on LDL Oxidation in Vitro The evidence supporting the hypothesis that LDL is the major atherogenic lipoprotein comes from epidemiological studies, clinical trials, studies in laboratory animals, heritable hypercholesterolemias, pathologic investigations, and studies in model systems. The role of oxidized LDL (oxLDL) in atherosclerosis, and consequently in CVD, is manifold. oxLDL induces cell adhesion molecule expression in aortic endothelial cells (60). Enhanced endothelial cell expression of chemotactic and adhesion molecules (i.e. E-selectin), ICAM-1 (CD54), and VCAM-1 (CD106), within the artery wall result in monocyte binding to endothelial cells, their entry into the vascular system, their differentiation into macrophages, and final conversion into foamy cells. oxLDLs also stimulate T-cells through the major histocompatability complex (MHC) and CD4+ helper T-cell receptor (61). Stimulated T-cells secrete: (i) IL-1 that increases smooth muscle cell proliferation, (ii) IL-2 that activates monocytes and increases T-cell proliferation, and (iii) IFN-γ that induces MHC expression in endothelial and smooth muscle cells. Activated macrophages also secrete a number of cytokines (TNF-α, TGF-β, M-CSF, G-CSF, PDGF) that influence the expression of mediators of endothelial activation, such as NO. Besides, the aldehydic products of LDL oxidation exert a direct toxic effect on the endothelium and platelets. To evaluate the potential of Chios Mastic to inhibit LDL oxidation in vitro, the method of Cu2+ induced oxidation was applied, using different volumes of Chios Mastic extracts, resuspended in ethanol (62). The effect of the polar extract from 2.5mg of Chios Mastic was almost equal to that of 5mg, while 50mg was shown to be the quantity required for the complete inhibition of LDL oxidation. For comparative purposes, a protection factor, expressed as % protection factor, is used. When fractionating to determine a structure-activity relationship, mastic oil, collofonium residue and both the acidic fractions of NaOH and Na2CO3 were found potent inhibitors of LDL oxidation. Acidic and neutral fractions were comparatively inactive (8.9±9.2% and 19.6±18.7% protection, respectively) Normal collection obtained by simply cutting the tree, exhibited remarkable protective activity against LDL oxidation (75.3±19.5% protection) while ‘liquid collection’ obtained by injecting phytohormones to the tree appeared to have considerably reduced activity 247 In Emerging Trends in Dietary Components for Preventing and Combating Disease; Patil, B., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.


(39.8±21.0% protection), indicating that the chemical constituents of Pistacia lentiscus from ‘normal’ and ‘liquid’ type collection differ. Furthermore, the pure triterpenes amyrin and oleanolic acid, which are natural constituents of Chios Mastic, exhibited relatively high protective activity (62.6±18.2% and 67.9±19.0% protection), however it did not exceed the overall activity of the crude product suggesting a synergistic effect between the active compounds. The isomers oleanolic and ursolic acids exhibited almost the same activity (67.9±19.0% vs 70.7±18.6 protection, respectively) indicating that structural differences do not influence the biological activity.

Effect on Peripheral Blood Mononuclear Cell (PBMC) Viability in Vitro During the early atherosclerotic process, monocytes differentiate into macrophages that themselves effect modifications in LDL, most importantly taking up the endothelial cell modified LDL. The uptake of oxLDL occurs via scavenger receptors of the class SR-A and SR-B, a member of which is CD36 scavenger receptor that binds to its lipid moiety (41). Macrophages become susceptible to apoptosis. Nucleus shrinks; organelles change; membrane loses integrity; DNA breaks down. Eventually macrophages are converted to foam cells, full of cholesterol and oxidized lipids. Macrophage foam cells form the early atherosclerotic lesions documented as the pathogenesis of cardiovascular heart disease (41). Also, cell death of T cells is abundant in human atherosclerotic lesions having consequences on the evolution of the atheroma. In an attempt to deepen our knowledge of the potential antiatherogenic and consequently cardioprotective effect of Chios Mastic, we employed a cell culture model of atherogenesis which included PBMC isolated from healthy volunteers and oxLDL (63). Oxidized LDL was found to be highly cytotoxic, resulting in severe damage of cultured cells, either apoptosis or necrosis, depending on the duration of exposure. The oxLDL toxicity on PBMC was almost minimized in the presence of Chios Mastic polar extract. As far as monocytes are concerned, loading with oxLDL significantly increased CD36 and upregulated CD36 mRNA expression. The significance of enhanced CD36 expression is due to the fact that once macrophages attract oxLDL to scavenger receptors they promote endocytosis and convert into cholesterol-loaded foam cells. Nutrients can influence gene expression directly or via gene promoters, via control of regulatory signals in nontranslated regions, and via post-transcriptional pathways. All the genes encoding proteins and the genes associated with transcriptional activation and signals modulating the transcription of the respective genes are potential candidates for gene–diet interaction. The capacity of dietary antioxidants to modulate gene expression has been investigated chiefly during the last decade. The antiatherogenic effect of Chios Mastic extract was evident attributed to the downregulation of CD36 both at the protein and at the transcriptional levels, to inhibition of both apoptosis and necrosis and restoration of GSH levels. The decrease of GSH (20.0%) in PBMC treated with oxLDL for 48h was similar to decrease (21.4%) in cells treated with the oxidizing agent for 72 h. The 248 In Emerging Trends in Dietary Components for Preventing and Combating Disease; Patil, B., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.


extract from Chios Mastic restored GSH levels utterly when treating cells under oxidative stress. Measurement of GSH levels after PBMC treatment individually with the polyphenolic and triterpenoid fractions revealed an increase 5.0% in GSH when treating cells with oxLDL and the polyphenolic fraction and 23.6% when treating cells with oxLDL and the triterpenoid fraction, compared to GSH measured when culturing cells with oxLDL alone. Consequently, triterpenes were the most active on the antioxidant defense of PBMC. Mass spectra has indicated oleanolic acid and urs-12-en-28-al as the major components of the triterpenoid fraction while indicated in the polyphenolic fraction were tyrosol, p-hydroxy-benzoic acid, p-hydroxy-phenylacetic acid, vanillic acid and traces of gallic and trans-cinnamic acids (64). Oleanolic acid and isomer ursolic acid have been found to increase glutathione and superoxide dismutase in Dahl salt-sensitive insulin resistant rat model of genetic hypertension (65). They have been also proven to exhibit antioxidant effect (62), antihyperlipidemic and antihypertensive effects and to prevent the development of atherosclerosis (66). Regarding polyphenols, these have been proven to be potent antioxidants (67). Further to the above, when evaluating the cardioprotective effect of Chios Mastic in humans, subjects ingesting high-dose Chios mastic powder exhibited a decrease in serum total cholesterol, LDL, total cholesterol/HDL ratio, lipoprotein (a), apolipoprotein A-1, apolipoprotein B (apoB/apoA-1 ratio did not change), SGOT, SGPT and gamma-GT levels. In subjects ingesting low-dose Chios mastic powder glucose levels decreased in males (68). The binding and recruitment of circulating monocytes to vascular endothelial cells are early steps in the development of inflammation and atherosclerosis, mediated through cell adhesion molecules that are expressed on the surface of endothelial cells. When evaluating the potential of the neutral extract of Chios Mastic to influence the expression of adhesion molecules and the attachment of monocytes in human aortic endothelial cells, inhibition of both the expression and attachment were observed, providing new insight into the beneficial effect of Chios Mastic on endothelial function (69). Immunomodulation using Chios Mastic. The Case of Inflammatory Bowel Disease (IBD) Regulation of the immune system includes a complex system of immune cells, pro-inflammatory and anti-inflammatory polypeptides and adhesion molecules. The Inflammatory Bowel Disease (IBD) refers to ulcerative colitis and Crohn’s disease. Incidence rates of 3 to 14 cases per 100,000 people are reported in the Western world (70), but pathogenesis remains unknown; genetic, environmental and immunologic factors seem to be responsible for IBD suffering, resulting in dysregulation of the immune system (71). Cytokines produced by immune cells contribute to the inflammatory response. Failure of controlling leukocyte recruitment enhances immune cell activation, which leads to further chemotaxis. Several inflammatory cytokines have been implicated in IBD and are elevated in colonic tissue and peripheral blood of IBD patients. TNF-α has been proven critical for IBD, but apart from TNF-α other cytokines play central roles in IBD. The proinflammatory cytokines IL-6 and IL-8 are produced in excess, while 249 In Emerging Trends in Dietary Components for Preventing and Combating Disease; Patil, B., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.


the immunosuppressive IL-10 is reduced in inflammed tissues. Both the excess of proinflammatory cytokines and the relative inefficiency of counterregulatory molecules are required for maintaining, amplifying, and perpetuating chronic inflammation in IBD (72). In addition, different cytokines induce the expression of adhesion molecules such as ICAM-1 on the endothelium, thus favouring the recruitment of new inflammatory cells. Imbalance between oxidant and anti-oxidant factors is also observed. In presence of inflammation reactive oxygen species inhibit antioxidant actions increasing lipid peroxidation. As a result, oxidative stress occurs. Corticosteroids, antibiotics and immunosuppressants are used to standardize symptoms (71). With increasing awareness of the harm of the long-term use of corticosteroids the use of natural compounds in treatment of autoimmune disorders attracts wider research and seems to be embraced by patients. When patients with Crohn’s disease were treated with Chios Mastic significant reduction in Disease Activity Index was reported (73, 74). Effectiveness was probably owed to significant decrease of the pro-inflammatory IL-6 (Table 3), inducing remission in seven out of ten patients. The importance of IL-6 in patients with Crohn’s disease has been well documented and mRNA for IL-6 is overexpressed in the inflamed mucosa of patients with active disease (72). Elevated IL-6 in plasma of patients with Crohn’s disease has been previously described (75). Because IL-6 is the main cytokine factor responsible for hepatic induction of acute phase proteins in Crohn’s disease, significant decrement in CRP was reasonable (Table 3). Oxidative stress has been proven to upregulate IL-6 gene expression. Chios Mastic treatment resulted in increase of plasma Total Antioxidant Potential (TAP) in Crohn’s disease patients (Table 3). Whether the antioxidant triterpenes and phenolics contained in Mastic act after being absorbed or act on the exposed gastrointestinal mucosa, remains uncertain. Although not as well absorbed as vitamins C and E, yet some phenolic compounds are absorbed, while those unabsorbed may remain in the lumen and become available for fermentation in the gut. Thus, gastrointestinal mucosa can be exposed to these compounds, or to their bacterial and systemic metabolites. Also, some triterpenes like glycyrrhetinic acid, the triterpene derivative of glycyrrhizin, have been shown to be bioactive in experimental gastric lesions. The anti-TNF-α treatment in TNF-mediated diseases, such as Crohn’s disease is developing. In active patients subjected to Chios Mastic treatment, secretion of TNF-α by isolated PBMCs showed significant decrease. This anti-TNF activity may be related to specific blockade of TNF-α secretion or to NF-κB pathway. It has been suggested that TNF-α regulates MCP-1 secretion via the activation of NF-κB (76). Yet, this was unlikely to occur in the case of Chios Mastic administration, given that MCP-1 concentration remained unaffected (Table 3). It is rather that the NF-κB pathway secondary to the decrease in TNF-α was not activated. A possible approach would be via the inhibition of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase. By blocking HMG-CoA reductase on human monocytes, cells reduce the production of TNF-α (77).

250 In Emerging Trends in Dietary Components for Preventing and Combating Disease; Patil, B., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.

Table 3. Effect of Chios Mastic oral administration in sera and PBMCs from patients with Crohn’s disease


Sera

Before

After

IL-6 (ng/mL)

0.02±0.01

0.007±0.003*

TNF-α (ng/mL)

0.03±0.01

0.01±0.005

MCP-1 (ng/mL)

0.1±0.04

0.07±0.02

CRP (mg/mL)

40.3±13.1

19.7±5.5*

TAP (mmol/L)

0.15±0.09

0.57±0.15*

Before

After

0.62±0.13

0.52±0.18

TNF-α (ng/mL)

2.1±0.9

0.5±0.4*

MCP-1 (ng/mL)

3.0±1.1

1.7±1.0

MIF (ng/mL)

1.2±0.4

2.5±0.7*

GSH (µmol/L)

34.0±15.8

56.6±10.3

PBMCs IL-6 (ng/mL)

* Asterisk points out statistically significant differences before and after treatment (P < 0.05).

Macrophage migration inhibitory factor (MIF) was originally described as an inhibitor of migration and chemotaxis of monocytes/macrophages. Suppressed secretion of MIF in mononuclear cells derived by patients with Crohn’s disease compared to the respective levels in healthy subjects indicates that monocytes are sensitized to chemotaxis. Increased secretion after treatment with Chios Mastic points to the inhibition of monocyte chemotaxis (Table 3). The significance of this is that migration of chemokine or peptide or nonpeptide stimulated monocytes and differentiation to macrophages into the site of inflammation is limited and inflammation is regulated. A steadily increasing number of research protocols has recently been developed and have contributed greatly to important advances in our current understanding of the immunological regulation by Chios Mastic. Regarding toxicity and acceptable dose, orally administered mastic of 521 mg/kg/day apparently enhanced preneoplastic lesion in a rat liver medium-term carcinogenesis bioassay, while 52.1 mg/kg/day was a non-promoting dose. The latter dose is almost equivalent to 2.9 g/day/person with the average human body weight regarded as 60kg. Most mastic doses reported in human studies are around this level, e.g. 5 g/day/person (68), 4 g/day/person (53), 2.2 g/day/person (73, 74), or 1 g/day/person (50). Favorable effects of mastic such as the anticarcinogenic potential could be achieved at relatively low doses without any toxicity (78, 79).



Greek Currants (Corinthian Raisins, Vitis vinifera L.) Currants (Corinthian raisins, Vitis vinifera L., Vitaceae Family) are dried vine fruits cultivated and processed since antiquity. Historical records confirm that grapes were dried under the sunlight since 1490 B.C. The climate in Greece was ideal for the growth of grapes, thus Greece became one of the main commercial markets for currants. The Greek vineyards produce different varieties of grapes, which are very competitive products in global market. Nowadays currnats represent about 8-10% of dried vine products production; other dried vine fruits varieties are the raisins – produced mainly in California and in some other mild climate countries – and the Sultanas – produced mostly in Turkey, Iran, Afganistan, South Africa, Australia, Chile, but also in Greece. Corinthian currants – produced from a special type of black grapes – are small sun-dried berries, coloured black to dark blue, and produced almost exclusively in Southern Greece. Based on the applied agricultural practices, the product properties, and the degree of product uniformity and cleanness, currants are classified in two main quality categories A and B. In the highest quality category A there are two subcategories, i.e. Vostizza and Gulf currants, the former considered as the superior quality one and holding a PDO name. Both Vostizza and Gulf currants are produced exclusively in North Peloponesse in the south of Greece while the Provincial currants (quality category B) are produced in Western Peloponesse and in two Ionian islands Zakynthos (Zante) and Cephalonia. Nutritionally, raisins are perfect alternative sweeteners, high in fiber, complex carbohydrates, and minerals and vitamins necessary for vitality. A portion of 40g contains usually 28-32g of sugar - mainly fructose -, 2g of fibers, very small quantities of protein (usually 1g), sodium (roughly 10mg), calcium and iron; its energy content is 110-140kcal. Greek currants content in potassium is very high – usually 310mg that corresponds to approximately 10% of daily need in humans. With respect to the dried vine products polyphenol content and antioxidant activity very limited literature data exist. In the study of Karadeniz et al. (80) the polyphenol composition of sun-dried, dipped, and golden raisins obtained from Thompson seedless grapes (Vitis Vinifera L. cv. sultanina) was reported. Chiou et al. (81) reported the content in total and simple individual polyphenols of the several currants subcategories, while their antioxidant capacity in terms of scavenging the DPPH• free radical was also assessed. Currants and Greek originated sultanas total polyphenol content was also reported by Kaliora et al. (82). Additionally in this latter study the in vitro antioxidant, antiatherogenic, and anticancer activity of the products was studied.

Antioxidant Activities of Currants The antioxidant capacity of currant methanol extracts as screened by the DPPH• radical scavenging assay was found similar for all three cultivars – Vostizza, Gulf, and Provincial – in the study of Chiou et al (81) while in the study of Kaliora et al (82) the decreasing order of scavenging activity was Gulf>Cretan sultanas>Provincial>Vostizza. 252 In Emerging Trends in Dietary Components for Preventing and Combating Disease; Patil, B., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.


Polar extracts from currants and sultanas inhibited the chemically-induced LDL oxidation and increased the levels of antioxidant GSH (82). oxLDL is associated with the pathogenesis of atherosclerosis, a key early stage of CVD, hence, the significance of this activity is evident. The effect on LDL was correlated to the polyphenol content, meaning that this effect could be attributed to the polyphenols as well. Polyphenols have been found to act against LDL oxidation, and their antioxidant capacity is related to their chemical structures. oxLDL is primarily responsible for GSH depletion creating an oxidizing environment required for γ-GSC induction and compensatory GSH synthesis (63, 83). A product from LDL oxidation namely tBHP, is responsible for GSH decrease in PBMCs. Glutathione (GSH) is modulated in disorders caused by free radical attack (84). Glutathione the most abundant antioxidant in cells is found predominantly in two redox forms: reduced and oxidized. Its protective action is based on the oxidation of the thiol group of its cysteine residue with the formation of GSSG, which in turn is catalytically reduced back to the thiol form by gluatathione reductase. Measurement of the GSSG level or determination of the GSH/GSSG ratio, is a useful indicator of oxidative stress and can be used to monitor the effectiveness of antioxidant intervention strategies. No difference was observed in intracellular GSSG with currant extract while elevated levels of total GSH was observed when pre-treating PBMC with the extracts (82). This shows that incubation of cells with the plant extracts possibly induces γ-glutamylcysteine synthetase. Ghibelli et al (85) have shown that GSH decrease occurs during cell death through a physiological process, i.e via physiological carriers responsible for GSH efflux. When inhibiting the carriers, they not only observed GSH restoration, but also reduced cell death, thus proving that decrease in GSH is an intrinsic part of cell death signalling, a necessary step to trigger the events of apoptosis. Exposure of cells to tBHP resulted in decrease of GSH and decrease in cell survival. Also, cell survival was found to be correlated to total polyphenol content and to antioxidant activity as to LDL oxidation, showing that inhibition of oxidative stress triggered cytotoxicity is owed to the antioxidant activity of the contained polyphenols. Because part of an agent’s potential to protect against atherosclerotic plaque formation may be due to the protection of cells from apoptosis, the protective effect of currant extracts on oxidative stress- induced apoptosis was observed under a fluorescence microscope. The cells exposed to oxidizing agent exhibited features of programmed cell death; however, cells that were pre-treated with the extract had reduced characteristics of apoptotic cells. Their morphological observation was similar to this of the control cells. These data, obtained from a non quantitative assay, suggest that the extracts inhibited oxidizing agent-induced apoptosis. Currants Anticancer Activity Gastric cancers, 90% of which are adenocarcinomas, constitute a major cause of mortality both in developed and developing countries, because currently available chemotherapeutic regimens are not very effective resulting in high recurrence rates and poor survival. Gastric cancer is recognized as a multifactorial disease. The pathogenesis of carcinogenesis embodies genetic factors, infection 253 In Emerging Trends in Dietary Components for Preventing and Combating Disease; Patil, B., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.


with Helicobacter pylori, environmental factors and lifestyle factors, including dietary habits. Preventive dietary strategies offer the best opportunities for control of the disease. To this end, research on natural products defensive against stomach cancer cell proliferation is of intense interest. High molecular weight fractions of tea have been found to induce apoptosis in stomach cancer MKN-45 cells (86). Resveratrol suppressed both synthesis of DNA and generation of endogenous O2but stimulated nitric oxide synthase (NOS) activity (87). Tea theaflavins have been shown to inhibit the growth of gastric cancer MKN-28 cells (88). Phenolic compounds isolated from sweetpotato leaves suppressed the proliferation of a stomach cancer cells Kato III (89). The methanol extracts obtained from currants of different origin in Greece and sultanas exhibited gastric cancer preventive efficacy by limiting cell proliferation and suppressing ICAM-1 levels, both at the protein and at the mRNA levels, in gastric adenocarcinoma cells (AGS cell line). Differences in the activity of different qualities of currants were probably due to both the geographic origins and the different qualities. Inter–group differences were statistically significant for total polyphenol content, meaning that total polyphenols differ in products of different geographical origin. The mechanisms of chemoprevention may be several including effects on cellular differentiation/cell cycle, apoptosis, and activation or deactivation of various enzyme systems such as the phase I or II biotransformation enzymes or antioxidant action. Protocatechuic acid and caffeic acid inhibited the growth of AGS cells through the induction of apoptosis associated with two signalling pathways; one was the activation of p38 signalling and the other was the stabilization of p53 (90). Currant and sultana extracts inhibited the proliferation of human gastric carcinoma cells through triggering apoptosis, rather than necrosis (82). A common feature of malignant cells is their ability to proliferate without restraint while apoptosis is a primary mechanism for the chemoprevention of cancer. Therefore, it is of interest to identify active compounds or mixtures of compounds from foods with apoptosis-inducing activity against cell lines. A causal relation between inflammation and cancer has been proposed by many research groups, suggesting that cancers arise at regions of chronic inflammation (91). In chronic inflammation, proinflammatory cytokines, such as TNF-a, are induced and activate transcription factors, such as NF-κB and activator protein-1 (AP-1). Transcription factors, in turn, modulate the levels of inflammatory cytokines, such as IL-8, as well as intercellular adhesion molecules, such as ICAM-1, which is highly expressed in the stomach. ICAM-1 protein and mRNA levels were decreased in the presence of currant extracts. ICAM-1 expression is dramatically increased at sites of inflammation, providing important means of regulating cell-cell interactions and thereby presumably inflammatory responses. Cancer cells with high ICAM-1 expression show a high invasive and metastatic potential along with the progression of the tumor.



In patients with gastritis gastric mucosal levels of IL-8 correlate with histological severity (92). In gastric cancer, IL-8 induces expression of adhesion molecules such as ICAM-1 (93). Expression of IL-8 gene is primarily controlled at the transcriptional level. NF-κB and AP-1 are inducible transcription factors and their binding sites are found in the promoter region of the IL-8 gene (94). In gastric cancer cell lines, synchronized action of NF-κB and AP-1 seems important in the expression of IL-8 in response to cytokine stimulation (95). Contrary, to decrease in ICAM-1 protein and mRNA levels, IL-8 protein or mRNA levels remained unchanged in the presence of currant extracts. ICAM-1 expression is regulated by NF-κB transcription factor solely (96). It is rather that the extracts regulate gastric inflammation via the decreased activation of NF-κB, but not of AP-1.

Polyphenols in Currants Chiou et al. (81) reported the total polyphenol content of the three currants sub-varieties – Vostizza, Gulf, and Provincial – in the range 155 - 246 mg GAE / 100g (mean 191±26 mg GAE/100g) with Provincial currants containing a rather higher total phenolic content than Vostizza and Gulf ones. Kaliora et al (82) have found similar total phenolic values for Vostizza and Provincial (from Messinia) currants; both these currants sub-varieties have been shown to contain considerably higher total phenolics than Sultanas obtained from the south of Greece (Cretan island). In this latter study Gulf currants have been reported to contain similar total phenolic content with that of Sultanas and considerably lower than the one reported by Chiou et al. (81). Grapes are a rich source of polyphenols (97) ranging from simple compounds to complex tannin-type substances. Phenolic acids, stilbene derivatives, and flavonoids are among the several classes of polyphenolic compounds present in grapes; grape polyphenols can also be found in the form of tartaric acid esters or glycosylated. With respect to the dried vine products Chiou et al. (81) studied currants content in simple polyphenols; data reported are summarized in Figure 4. The polyphenolic profile of the several currant sub-varieties was not differentiated, with the exception of the absence of 3,4-di-hydroxy-phenylacetic acid from the Vostizza samples. Seventeen polyphenol species were identified in Gulf and Provincial currants and 16 in Vostizza ones. Total simple polyphenols ranged from 4.81±0.99 mg /100 g in Vostizza to 6.71±2.03 mg /100 g in Provincial currants (mean 5.60±1.00 mg /100 g). In all studied samples benzoic acids predominated, while among them vanillic acid was the main polyphenol species in all cases. Resveratrol – a stilbene with anticarcinogenic properties – was present at mean concentration 0.19±0.07 mg / 100 g. Though not quantified, the presence of catechin and epicatechin and the triterpenic oleanolic acid was also reported.



Figure 4. Benzoic acids (vanillic acid, gallic acid, syringic acid, p-hydroxybenzoic acid, protocatechuic acid), cinnamic acids (cinnamic acid, phloretic acid, p-coumaric acid, ferulic acid, caffeic acid), phenylacetic acids (p-hydroxy-phenylacetic acid, 3,4-di-hydroxyphenylacetic acid), phenols (tyrosol, vanillin), flavonols (quercetin, kaempferol) and stilbenes (resveratrol) present in the three currants sub-varieties.

Conclusions The principal aspects of the most healthful nutritional recommendation namely Mediteranean Diet include among others high consumption of legumes, fruits, as well as medicinal plants such as herbs or Chios Mastic. Legumes are particularly characteristic of the Mediterranean diet and their nutritional value is considered very high, while additionally identification of different phenolic and terpenic compounds in them highlights their potent role as health promoting food components. Herbs infusions are rich in polyphenolic compounds which together with their essential oils’ components are expected to exert beneficial action towards several denegerative diseases. Mastic is widely used as a food additive and exerts beneficial health effects attributed to several bioactive constituents. The cardioprotective and immunoregulating effects established in vitro and in vivo need to be further studied in larger cohorts, and may aid to design new therapies for prevention in atherosclerosis and other related cardiovascular diseases, as well as in prevention in inflammatory bowel diseases. The in vitro antioxidant/antiatherogenic effectiveness of currants, owed mainly to the contained polyphenols, and their chemopreventive effect are of immense interest in the development of strategies to prevent or delay cardiovascular events and several forms of cancer. In future, more than just nutritional items, such natural products may be considered as factors with priorities in medical applications. 256 In Emerging Trends in Dietary Components for Preventing and Combating Disease; Patil, B., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.


List of Abbreviations AAE AP-1 CD36 CRP DNA DPPH• FRAP GAE G-CSF γ-GSC GSH GSSG ICAM-1 IFN-γ ILLDL MCP-1 M-CSF MIF mRNA NF-κB NO PBMC PDGF SGOT SGTP TAP TBARS tBHP TE TGF-β TNF-α VCAM-1

Ascorbic acid equivalents Activator protein-1 Cluster of Differentiation 36 C-Reactive protein Deoxyribonucleic acid 2,2-Diphenyl-1-picrylhydrazyl free radical Ferric ion reducing antioxidant power Gallic acid equivalents Granulocyte-colony stimulating factor γ-Glutamyl cysteine synthetase Glutathione Glutathione disulfide Inter- Cellular Adhesion Molecule-1 Interferon-gamma InterleukinLow density lipoprotein Monocyte chemoattractant protein-1 Macrophage colony stimulating factor Macrophage migration inhibitory factor Messenger Ribonucleic acid Nuclear factor-kappa Beta Nitric oxide Peripheral Blood Mononuclear Cells Platelet-derived growth factor Serum glutamic oxaloacetic transaminase Serum glutamic pyruvic transaminase Total antioxidant potential Thiobarbituric acid tert-Butyl hydroperoxide Trolox equivalents Transforming growth factor-beta Tumor necrosis factor-alpha Vascular Cell Adhesion Molecule-1

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